Long non-coding RNA-NONMMMUT004552.2 regulates the unloading-induced bone loss through the miRNA-15b-5p/Syne1 in mice

Exercise-induced mechanical loading can increase bone strength whilst mechanical unloading enhances bone-loss. Here, we investigated the role of lncRNA NONMMUT004552.2 in unloading-induced bone-loss. Knockout of lncRNA NONMMUT004552.2 in hindlimb-unloaded mice caused an increase in the bone formation and osteoblast activity. The silencing of lncRNA NONMMUT004552.2 also decreased the osteoblast apoptosis and expression of Bax and cleaved caspase-3, increased Bcl-2 protein expression in MC3T3-E1 cells. Mechanistic investigations demonstrated that NONMMUT004552.2 functions as a competing endogenous RNA (ceRNA) to facilitate the protein expression of spectrin repeat containing, nuclear envelope 1 (Syne1) by competitively binding miR-15b-5p and subsequently inhibits the osteoblast differentiation and bone formation in the microgravity unloading environment. These data highlight the importance of the lncRNA NONMMUT004552.2/miR-15b-5p/Syne1 axis for the treatment of osteoporosis.

microgravity unloading, there was a highly expressed lncRNA (lncRNA NONMMUT004552.2) related to bone metabolism, and at the same time, we also found a highly expressed gene (Syne1).However, whether NON-MMUT004552.2 and Syne1 in the osteogenic region can regulate bone loss after mechanical unloading, and how they play their roles still need to be further explored.
In this study, we investigated the function of lncRNA NON-MMUT004552.2(Table S1) in HU mice, and confirmed that lncRNA NONMMUT004552.2 decreased the bone formation and osteoblast activity in HU mice.lncRNA NONMMUT004552.2 could promote the osteoblast apoptosis and inhibit osteoblast mineralization in vitro.In addition, The lncRNA NONMMUT004552.2 could regulate Syne1 expression by interacting with miR-15b-5p.The lncRNA NONMMUT004552.2 partly promoted apoptosis and reduced differentiation in MC3T3-E1 cells in a manner partially dependent on miR-15b-5p and Syne1 in a microgravity unloading environment.These findings highlight the lncRNA NON-MMUT004552.2as a therapeutic target for the treatment of osteoporosis and pathological osteopenia.

Results
Knockout of lncRNA NONMMUT004552.2 alleviates the loss of osteoblast activity and bone formation in hindlimb-unloaded (HU) mice To identify the mechanosensitive lncRNAs in osteoblasts, we performed RNA sequencing of the primary osteoblasts from mice exposed to simulated microgravity (MG) unloading conditions.We found that lncRNA NON-MMUT004552.2(P < 0.01) and Syne1 (P < 0.05) were highly expressed by qRT-PCR in HU mice compared with control group (Figs.1A and 1B).To further investigate how they interact in bone metabolism, hindlimbunloaded (HU) mouse models are the most commonly used in vivo models for inducing bone loss due to unloading.After 21 days of hindlimb unloading, a marked increase in the number of TUNEL+ cells were observed in the mice distal femurs of the HU group compared to the control group.Fewer apoptotic cells were observed in the HU+si-NON-MMUT004552.2 group compared to HU + si-NC mice (P < 0.001, Fig. 1C).The number of Bglap+ osteoblasts were significantly lower in mouse femurs of the HU group compared to the control group.Bglap+osteoblasts in the HU+si-NONMMUT004552.2 group were higher than HU+si-NC mice (P < 0.001, Fig. 1D).Compared to the control group, HU mice showed a reduced total bone area following H&E analysis.The administration of si-NONMMUT004552.2significantly restored the bone area per total area (P < 0.001, Fig. 1E).Bone formation (the mineral apposition rate (MAR)) was markedly lower in the HU group compared to control mice.Bone formation was higher in the si-NONMMUT004552.2-treatedHU groups compared to HU + si-NC mice (P < 0.001, Fig. 1F).Collectively, these data show that lncRNA NONMMUT004552.2 facilitates the loss of bone formation and osteoblast activity in HU mice.

Discussion
Bone loss is regulated by various cytokines, hormones and miRNAs 2,24 .However, the role of lncRNAs in unloading-induced bone loss is less well understood.In this study, we reveal a mechanism by which the lncRNA NONMMUT004552.2 inhibits the osteoblast differentiation and promotes the bone loss following mechanical unloading in mice.The silencing of lncRNA NONMMUT004552.2 was found to inhibit apoptosis, promote osteoblast mineralization in MC3T3-E1 osteoblast.we further confirmed that the lncRNA NONMMUT004552.2 can increase the apoptosis and decrease the mineralization of MC3T3-E1 cells in the MG unloading environment, and that it can regulate Syne1 expression in a manner partially dependent on miR-15b-5p.This highlights the lncRNA NONMMUT004552.2/miR-15b-5p/Syne1 axis as a therapeutic target during osteoporosis.
Osteoblasts are key mediators of bone formation, responsible for the synthesis, secretion and mineralization of the bone matrix, allowing bone to grow and rebuild 25 .Increased osteoblast differentiation and the prevention of osteoblast apoptosis are important mechanisms in the pathogenesis of osteoporosis 26,27 .Multiple lncRNAs regulate bone formation and osteoblast differentiation 28 .LncRNA MEG3 activates the transcription of BMP4 through the disassociation of SOX2, which promotes osteoblast differentiation in MSC 29 .LncRNA Bmncr alleviates fat accumulation in bone marrow cells and prevents bone loss by facilitating the assembly of the RUNX2/PPARG and the TAZ transcriptional complex during aging 30 .LncRNA PGC1β-OT1 stimulates progenitor cell osteoblast differentiation, whilst LncRNA OGRU promotes bone formation 2 .To date, lncRNAs and circular RNAs, have been thoroughly characterized as ceRNAs for microRNA  binding 31 .Here, we show that lncRNA NONMMUT004552.2 localizes to the cytoplasm of osteoblasts and promotes unloading-associated bone loss through its interaction with miR-15b-5p.
Previously, it has been reported that miR-15b-5p is a a positive regulator for osteoblast differentiation, In human BMSCs, miR-15b promotes the osteoblasts differentiation and shows a high amount of ALP and type I collagen.This process targets Smurf1 which protects Runx2 from degradation 23 .In addition, The expression level of miR-15b is also regarded as one of the criteria for the osteogenic evaluation 32 .In this study, the experiments strongly suggested that miR-15b-5p is unloading-sensitive, and that its levels can be decreased by lncRNA NONMMUT004552.2 during unloading.Furthermore, we demonstrated that Syne1 is the target of miR-15b-5p.
Syne1, located on chromosome 6, encodes a spectrin repeat containing protein expressed in skeletal and smooth muscle, and peripheral blood lymphocytes, that localizes to the nuclear membrane 33,34 .It plays a role in cardiomyocyte and skeletal muscle development, particularly in DNA damage response pathways 35 .The SYNE1 expression also impacted stem cell pluripotency and differentiation capacity 36 .Most importantly, inhibiting the expression of Syne1 in rat mesenchymal stem cells decreases cell proliferation and increases apoptosis 37 .However, in the present study, we found that Syne1 had highly expressed in the HU mice.Interestingly, lncRNA NON-MMUT004552.2and Syne1 show the same expression patterns.Additionally, TargetScan and luciferase activity assays confirmed the binding between Syne1 and miR-15b-5p.Moreover, Syne1 expression was down-regulated by miR-15b-5p.miR-15b-5p inhibitor or Syne1 overexpression reduced the upregulation of osteoblast differentiation induced by the si-NONMMUT004552.2 in unloading condition.Thus, our results revealed that lncRNA NONMMUT004552.2 promotes bone loss by sponging miR-15b-5p, which subsequently regulates Syne1 expression.
In summary, our findings show that lncRNA NONMMUT004552.2 increases the apoptosis and inhibits osteoblasts differentiation during mechanical unloading-induced bone loss.lncRNA NONMMUT004552.2, which is a critical regulator of osteoblast function, and promotes the bone loss by functioning as a miR-15b-5p sponge to stimulate Syne1 expression.represents the important therapeutic target for osteoporosis in response to mechanical loading.

Methods
HU mouse models C57BL/6 J mice (6-month-old; Male) were purchased from the Beijing Vital River Laboratory (Beijing, China) and housed in a 21 °C incubator with a 12 h light/dark cycle and free access to food and water.Mice were were randomly divided into the following groups (n = 6 per-group): [1] Control; [2] HU; [3] HU+si-NONMMUT004552.2; [4] HU+si-NC.The hindlimb unloading (HU) model is one of the models of bone loss caused by mechanical unloading as previously reported 14,38 .Briefly, The mouse's tail was attached to a piece of surgical tape that formed a loop close to the end of the tail.Three strips of elastic adhesive bandage were used to secure the surgical tape that was placed on the tail.The mouse's forelimbs had full access to the entire cage.HU mice were hung from the top of the cage by the tail at a 30°angle with only the forelimbs touching the floor for 4 weeks, which allowed them to move and access food and water freely.The control group was mice without suspended tails in cage.Before HU, we encapsulated an osteogenic siRNA that target lncRNA NONMMUT004552.2 (si-NONMMUT004552.2) to the bone formation zone, mice in the experimental group were injected with si-NC, and si-NONMMUT004552.2plasmids via the caudal vein with 2 mg kg −1 plasmids every day for 3 consecutive days using the (AspSerSer) 6-liposome delivery system 9,19 .After 3-weeks of tail suspension, mice were euthanized and the bilateral femurs and tibiae harvested.No mice died during the procedure.All protocols were approved by the Animal Ethics Committee guidelines of Beijing Viewsolid Biotechnology Co. Ltd. (VS2602A12318).

TUNEL assays
Tibias from HU mice were fixed in 4% paraformaldehyde, decalcified, paraffin embedded and stained using the DeadEnd™ Fluorometric TUNEL System.Then, slides were stained with the DeadEnd™ Fluorometric TUNEL System (Promega, USA), and imaged using a fluorescent microscope (Evos FL Cell Imaging System; Life Technologies).DNase treatments were used as positive controls (green) for TUNEL staining.

Immuno-and histological analysis
Femurs from HU mice were fixed in 4% paraformaldehyde, decalcified using 10% ethylenediaminetetraacetic acid (EDTA, Beyotime Biotechnology, Shanghai, China) and embedded in paraffin.For immunohistochemistry, sections were dewaxed, blocked in 5% goat serum and stained for 24 h at 4 °C with primary antibody against the following specific protein Bglap (1:50; ab93876, Abcam).Subsequently, diaminobenzidine and hematoxylin were used to detect immunoreactivity.For histological analysis, Sections were stained with H & E and detected using diaminobenzidineused. Slides were imaged on a FV1000 confocal microscope (Olympus, Japan).

Double calcein labeling assay
To evaluate the dynamic indexes of bone formation, mice were intraperitoneally injected with 8 mg/kg calcein (body weight, Sigma, St. Louis, MO, USA) 10 days and 3 days before sacrifice.Fibias were harvested, fixed in 4% paraformaldehyde for 2 days and embedded in polymethylacrylate.Samples were cut into 3 sections (thickness ~50 μm) using a hard tissue slicing machine (SP1600, Leica, Germany) in the dark.Slides were imaged following double-calcein labeling by confocal microscopy (LSM800, ZEISS, Germany).The distance between two fluorescence-labeled lines as measured three times with Image J software was used to evaluate the mineral apposition rate (MAR) of bone.

Cell culture
Mouse MC3T3-E1 osteoblasts were purchased from the Chinese Academy of Sciences (China).Cells were cultured at 5% CO 2 , 95% humidity at 37 °C in α-MEM supplemented with 10% FBS and 1% penicillin or streptomycin.Cells at passage 8-12 were induced with osteogenic media containing 10 mM β-glycerophosphate, 100 nM dexamethasone and 50 μM ascorbic acid.Assayed were performed on a minimum of 3 occasions.

Cell culture under microgravity (MG) unloading condition
A two-dimensional (2D) clinostat was used to simulate the effects of MG for cells cultured.Briefly, MC3T3-E1 cells (1 × 10 5 cells per well) were plated on the cover glasses in a 6-well plate.After ~8 h, cells adhered to the walls, and the cover glasses were inserted into a chamber filled with culture medium.The distance between the cover glasses and the rotating axis of the chamber was 12.5 mm.Then, the caps of the chamber were tightened after all bubbles were gently removed.Finally, the chambers were placed into a clinostat and rotated around a horizontal axis at 24 rpm.The vertical rotation group served as the control group.The whole process of cell culture in the MG unloading environment was carried out at 37 °C.Experiments were carried out according to the previous research 9 .
Cell counting kit-8 (CCK8) assay CCK-8 assay was performed to evaluate the viability of osteoblasts.MC3T3-E1 cells were plated in 96-well plates at a density of 5 × 10 3 cells per well.After 24, 48, 72, and 96 h respectively, CCK-8 solution (10 µL, Dojindo, Kumamoto, Japan) was added into each well and the plates were incubated at 37 °C for 2 h.The absorbance value of each well was detected by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm wave length.

Flow cytometry
The transfected MC3T3-E1 cells were detached using trypsin (0.125%) and resuspended with phosphate-buffered saline (PBS).Cells were pelleted at 1000 rpm for 5 min and stained with Annexin V-FITC Apoptosis Detection Kits (BioVision, USA).Cell pellet was resuspended in 200 mL annexin V binding buffer, and the cells were counter-stained with 5 μL propidium iodide (PI) before analysis.Apoptotic cells were quantified by flow cytometry (BD Bioscience, USA), and the data were analyzed with Cell Quest software (Becton Dickinson, San Jose, USA).
Alizarin red staining MC3T3-E1 cells were seeded into 12-well plates and cultured in osteogenic medium for 21 days.Cells were fixed in 70% ethanol on ice for 1 h, washed in ddH 2 O and stained with 40 mM Alizarin red S (Sigma-Aldrich, Missouri, USA) at a pH = 4.2 for 15 min.Cells were washed in ddH 2 O and PBS.Representative images were acquired using a confocal microscope (Olympus BX51) equipped with a digital camera (C2si confocal microscope, Nikon Corporation).To quantify the calcified matrix, 200 μL of 5% perchloric acid (Cat.244252; Sigma-Aldrich) was added to each well and absorbance was measured at 420 nm using a microplate spectrophotometer.

ALP activity and labeling
Supernatants from MC3T3-E1 cells were centrifuged at 12,000 rpm for 15 min and ALP activity measured using commercial kit (ALP assay kit: Nanjing Jiancheng Technological Inc., China).Values were normalized to protein content through BCA protein assay kit (Thermo Fisher Scientific, USA).For ALP staining, cells were fixed with 4% paraformaldehyde (Sigma, Shanghai, China) for 15 min at room temperature and washed three times with phosphate-buffered saline (PBS).ALP was labeled in fixed cells using NBT/BCIP staining kits (Beyotime Biotechnology, China).Cells were imaged on a digital camera (Coolpix 4500, Nikon, Tokyo, Japan).

Statistical analysis
Data are expressed as the mean ± SD.Statistical analysis was performed using GraphPad Prism 7.0.All data for cell analysis were generated from three independent replicates.For animal analyses, six mice were assigned to each experimental group.Prior to the statistical analysis, the comparison data were normally distributed.Single group comparisons were performed using a Student's t-test.Multiple group comparisons were performed using two-way ANOVA or one-way ANOVA followed by Tukey's posthoc test.P < 0.05 was deemed significant.
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Fig. 1 |
Fig. 1 | Knockout of lncRNA NONMMUT004552.2 counteract the loss of bone formation in hindlimb-unloaded (HU) mice.A qRT-PCR analysis of LncRNA NONMMUT004552.2 level in HU mice.B qRT-PCR analysis of Syne1 level in HU mice.C Representative TUNEL images of the distal femurs in the indicated groups.TUNEL staining of apoptotic cells is green, whereas DAPI staining is blue.Scale bar = 50 µm; N = 6.D Representative images of Bglap staining of the distal femurs of